Chromosomal fragile sites are specific regions of mammalian chromosomes that are exceptionally sensitive to formation of chromosome gaps and breaks when cellular DNA replication patterns are perturbed. The molecular basis of fragile sites is of considerable interest. However, only two fragile sites have been cloned: FRAXA and FRAXE. Both are X-linked and are associated with mental retardation and expansion of a CCG repeat in the 5'portion of genes. The most sensitive of all fragile sites in the human genome is the constitutive fragile site FRA3B at 3p14.2. The isolation and characterization of this fragile site is the focus of this study. Over the past 3 years, we have mapped a series of YAC clones in the 3p14.2 region resulting in the identification of a YAC spanning both FRA3B and the t(3;8) breakpoint associated with hereditary renal cell carcinoma. We have isolated phage subclones from this YAC and used these in FISH experiments to further resolve the position of the fragile site. We have also constructed somatic cell hybrids with deletions, translocations and neo r- plasmid integrations in the region which may serve as markers for the fragile site. Sequence analysis and screening for repetitive DNA have been initiated. We will now further characterize and expand upon the nearly complete contig of genomic subclones isolated in the FRA3B region to conclusively identify the DNA sequences responsible for fragile site formation. To accomplish this goal, the contig of phage/cosmid clones across the FRA3B region will be completed and clones crossing the fragile site identified by FISH on cells expressing the fragile site. Clones spanning translocation/deletion breakpoints and neo-plasmid integrations at or near FRA3B will also be identified. The contig will be analyzed for tri- and tetranucleotide repeats and individual clones used in transfection experiments to determine the smallest region necessary for fragile site formation. Candidate clones or subclones of highest interest will be sequenced, and all repetitive DNA will be analyzed for polymorphisms in the normal population. Failure to identify repetitive DNA associated with FRA3B may indicate that higher order processes of replication and gene expression are responsible for fragile site formation. To this end, the replication timing of genomic DNA surrounding FRA3B will be investigated. The fragile site region will be analyzed for the presence of genes whose expression might be affected by fragile site expression, or correlated with the timing of fragile site replication. This study will address questions regarding the molecular nature of common fragile sites, their effect on chromosome structure and their potential mechanistic role in genome instability and chromosome breakage.